Understanding the Interaction Between Anabaena Sp. and a Heterocyst-Specific Epibiont
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Understanding the interaction between Anabaena sp. and a heterocyst-specific epibiont Iglika V. Pavlova MBL Microbial Diversity course 2005 Abstract Anabaena sp. and an associated heterocyst-specific epibiontic bacterium were isolated from School Street Marsh in Woods Hole, MA in 1997 during the Microbial Diversity course. A two-member culture of the cyanobacterium and the epibiont have been maintained at WHOI by John Waterbury. Anabaena species with similar heterocyst-specific epibionts have also been isolated from other locations (7, 8, 9). Successful culture of the epibiont separately from the School Street Marsh cyanobacterium was achieved on two occasions (1, 10), but the isolates were not preserved. The epibiont was identified to be an α-proteobacterium within the Rhizobiaceae group (10). The close and specific association between the cyanobacterium and the epibiont strongly suggests there might be a physiological benefit or benefits to the cyanobacterium, the epibiont, or to both partners. This study was designed to understand the potential benefits of the interaction for the epibiontic bacterium. Several approaches were used to isolate the epibiont in pure culture, unsuccessfully. This report describes the rationale for undertaking the project and the approaches used to isolate the epibiont, in the hopes that this will be useful information for the future isolation of an axenic epibiont culture. Rationale Cyanobacterial associations with heterotrophic bacteria from environmental isolates have been described before and have been implicated in increasing the growth of the cyanobacterium (5, 6, 7). Benefit to the cyanobacterium may be derived from a range of associations, including the presence of heterotrophic bacteria in the culture medium, attraction of such bacteria to cyanobacterial secretions (either along the whole filament, or secretions stemming from the heterocyst in filamentous bacteria), or the attachment of the bacterium to the cyanobacterium outer surface (5, 6, 7, 9). The attachment of bacteria to the outer surface, and the specific attachment to heterocysts to the exclusion of vegetative cells, is very interesting as the closeness and specificity of the interaction indicates that it may have been selected for over time, with a benefit to one or both partners. The specificity of epibiont attachment is not under question, as the epibiont has not been observed to bind to vegetative cells. Importantly, the epibiontic bacterium was shown to associate de novo to heterocysts that are very likely to be non-metabolizing (10). The issue regarding this two-species interaction is whether the specificity is based on metabolic benefits in addition to the specific attachment. The epibiont side of the interaction, while more likely to involve benefits than the cyanobacterial side (i.e., a parasitic interaction is more likely for the epibiont than for the cyanobacterium), has not been investigated and was the focus of this proposal. A specific association of epibiont to heterocyst based on metabolism is not hard to envision, as heterocyst cells are physiologically distinct from vegetative cells. Heterocysts are the site of nitrogen fixation in filamentous cyanobacteria that harbor them (3). Moreover, they are thought to have evolved as a result of selection for a specialized low-oxygen environment for nitrogen fixation, an extremely oxygen-sensitive process. One of these specializations is a thick cell wall, with a specific lipopolysaccharide (LPS) on the surface of the cell, which can serve as a site for attachment and as a source of nutrients for the epibiont (3). The Anabaena LPS contains glucose, galactose and mannose (12). Nitrogen fixation in the heterocyst provides other possible nutrients for the epibiont. First, ammonia is generated, which through a series of intermediates (e.g., glutamine) is converted to phycocyanin. Phycocyanin is a polypeptide made up of repeating units of arginine and aspartate, which serves for nitrogen storage and transport. At the ends of the heterocyst, the degradation products of phycocyanin, arginine and aspartate, are found. Second, nitrogenase produces as a co-product molecular hydrogen, which is an attractive electron donor for many bacteria, and could be used by the epibiont. Importantly, there is some preliminary evidence that hydrogen may play a role in the Rhizobium-legume symbiosis. Many such symbioses harbor Rhizobium strains that are defective in uptake hydrogenase (HUP), an enzyme that oxidizes hydrogen with energy conservation, and hydrogen is found to leak out of the nodules (2, 11). We currently don’t know whether the School Street Marsh Anabaena sp. has HUP, and if it does, how much of the molecular hydrogen is metabolized by this enzyme. Based on the above considerations, the following two major experimental approaches were designed to address the potential benefits for the epibiont of the association with the cyanobacterium: 1. Determine growth requirements of epibiont Currently, the epibiont has only been grown in complex rich media (MP media, see recipe below). One approach is to test different sources of carbon and nitrogen, in the presence or absence of hydrogen gas, and measure biomass accumulation. A second approach is to do chemotaxis assays to analyze epibiont chemoattraction to different sources of carbon and nitrogen, and to hydrogen gas. 2. Quantitative electron microscopic studies of de novo association and dissociation between epibiont and Anabaena sp. For the association experiments, the two axenic cultures will be mixed in different media, and the following parameters measured over time using electron microscopy: a) the number of heterocysts harboring epibionts, b) the number of epibionts attached to each heterocyst, c) the number of epibionts not attached to the Anabaena sp., and d) whether any vegetative cells have epibionts attached to them. For the disassociation experiments, a growing two-membered culture will be monitored over time for the association between epibionts and newly formed heterocysts in various media. The prediction is that if the epibiont attaches to obtain hydrogen and/or carbon sources from the cyanobacterium, it will not attach when these are available in the environment. Scanning Electron Microscopy 1. Cyanobacterial filaments were placed onto a glass slide with appropriate medium, and the clumps were teased apart gently and placed onto a 0.2 µm filter. 2. Samples were fixed for 4 hours in 2% glutaraldehyde, 1.5% formaldehyde solution. 3. Samples were dehydrated with 2,2-dimethoxypropane (only one was sufficient) and kept in absolute ethanol overnight. 4. The next day, the samples were dried using a critical-point drier, mounted on an SEM stub and sputter-coated, and stored in a dessicator. 5. SEM was performed on a JEOL JSM-840 scanning electron microscope. Figures 1-4 show some of the photomicrographs obtained. Figure 1 shows a cyanobacterial clump. Figures 2-4 are close-ups of single filaments with heterocysts and attached epibionts. Note the range in length of the filaments from ~1 to ~8 µm. Approaches for the isolation of an epibiont culture Media Both liquid and solid media was used for the purifications. The first medium described below is for culture of cyanobacteria; the other media are heterotrophic media used for the isolation of the epibiont. All recipes are for 1 L of medium. All media used had 25% seawater, which is the same salinity as the media used for the two-member culture. All solid media were with 1% agar. BOX and BNAX media These are the media that John Waterbury routinely uses in his lab for culture of cyanobacteria. Since I was using modular media (different combinations), I made stock solutions and would mix them as desired, which is a different approach from how this media is typically prepared. There is no need to adjust pH, as the seawater acts as a very efficient buffer. BOX (ml) BNAX (ml) Cyano trace metals (g/L) ddH20 750 750 ZnS04x7H20 0.222 Filtered seawater 250 250 MnCl2x4H20 1.4 20 mM Na2C03 0.1 0.1 Co(N03)2x6H20 0.025 20 mM K2HP04 0.1 0.1 Na2MoO4x2H20 0.39 200 mM Na2N03 -- 1.0 Citric Acid hydrate 6.25 200 mM NH4Cl -- 0.1 Ferric Ammonium Cyano trace metals 0.5 0.5 Citrate (brown) 6.0 0.1 mM EDTA (disodium) 0.5 0.5 Oligotrophic CYPS medium Many bacteria grow optimally under limiting amounts of nutrients. I also thought that this media could provide a way to promote the growth of the epibiont and discourage the growth of the contaminating bacteria (see below, “Major problem: culture contamination”). The CYPS medium is commonly used by Jeanne Pointdexter for the growth of oligotrophic marine organisms. Casamino acids 0.05% Peptone 0.05% Yeast extract 0.05% Seawater 25% Marine Purity medium This is the medium that was used successfully for the purification of the epibiont (10). Please note that this medium does not necessarily have the optimal concentrations and combinations of nutrients for epibiont growth, as indicated by the slow growth of the epibiont under these conditions (10, John Waterbury, personal communication). MP medium (g) NaCl 20 Make separately NaCl/AC broth, MgS04, and CaCl2 solutions 0 AC broth* 17 with dH20, autoclave, cool down to <65 C, and mix. MgS04x7H20 8 CaCl2x2H20 0.6 *The manual directions for AC broth (Difco) is 34 g/L. Brad Stevenson (10) used 17 g/L. I used both 17g/L and 8.5 g/L. Other media combinations Other combinations of oligotrophic media were also used to better define the growth requirements of the epibiontic bacterium and to provide (as above) a selection against the contaminating bacteria. The major principle was to use a base medium and provide different carbon sources: Base medium Carbon sources (additional) BOX medium components All at 0.05% 0.05% peptone Mannose, malate, glucose, galactose, cellobiose, acetate, succinate Combinations Base medium + one carbon source Other culture parameters Cyanobacterial cultures were grown in a 300C incubator with a 12 h light/12 h dark cycle, with no shaking.